Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum

Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum (http://www.molecularstation.com/forum/)
-   RNA Techniques Forum (http://www.molecularstation.com/forum/rna-techniques-forum/)
-   -   Will DEPC interfere with PCR reaction (http://www.molecularstation.com/forum/rna-techniques-forum/3561-will-depc-interfere-pcr-reaction.html)

jgleema 07-11-2008 12:13 PM

Will DEPC interfere with PCR reaction
 
Hi,
I isolated RNA, and in the steps involved used DEPC treated water. But while using that in RT-PCR, will DEPC interfere with the reaction? Is that necessary to aliquot the primers also in DEPC treated water?

admin 07-11-2008 05:53 PM

Re: Will DEPC interfere with PCR reaction
 
Hello Jgeleema,
have you inactivated DEPC/Rnases with autoclaving?

DEPC should be ok in your PCR after dilution, but MiliQ water or RNAse Free water should be better than DEPC treated water as DEPC only inactivates RNases without removing them.

Some inactivated Rnase can remain in solution and be activated after DEPC is damaged/removed.

Excess DEPC is destroyed by autoclaving to 121 C however it is hydrolyzed into pyrocarbonate and carbon dioxide. Ethanol is also generated as a side-product which although evaporates from solution may inhibit your PCR reaction a bit.

Some side products remain still after autoclaving and spent reagent contributes significantly to the water's total organic carbon (TOC) content.

jgleema 07-14-2008 06:44 AM

Re: Will DEPC interfere with PCR reaction
 
Hi admin,
Thank you for your reply. It was very useful to me.
Thank you.

navidagar 06-09-2010 04:07 AM

Re: Will DEPC interfere with PCR reaction
 
Hi !
i'm just strted working with rna wants to know about the depc treatment and if it is possible to work without it.

Jon Moulton 06-09-2010 03:18 PM

Re: Will DEPC interfere with PCR reaction
 
DEPC reacts with nucleic acid bases, alkylating them. It is best to autoclave water after DEPC treatment, as this will decompose the unreacted DEPC.

Henderson, R.E., Kirkegaard, L.H., and Leonard, N.J. 1973. Reaction of diethylpyrocarbonate with nucleic acid components. Adenosine-containing nucleotides and dinucleoside phosphates. Biochim. Biophys. Acta 294:356-364.


All times are GMT. The time now is 01:50 PM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved

Page generated in 0.09055 seconds with 11 queries